Oncogenic and Rasopathy-Associated K-RAS Mutations Relieve Membrane-Dependent Occlusion of the Effector-Binding Site

Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site

Mohammad T. Mazhab-Jafaria,1, Christopher B. Marshalla, Matthew J. Smitha, Geneviève M. C. Gasmi-Seabrooka, Peter B. Stathopulosa,2, Fuyuhiko Inagakib, Lewis E. Kayc,d, Benjamin G. Neela, and Mitsuhiko Ikuraa,3

aDepartment of Medical Biophysics, Campbell Family Cancer Research Institute, Princess Margaret Cancer Centre, University of Toronto, Toronto, ON, Canada M5G 2M9; bFaculty of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan; cDepartments of Molecular Genetics, Biochemistry, and Chemistry, University of Toronto, Toronto, ON, Canada M5S 1A8; and dProgram in Molecular Structure and Function, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8

Edited by Thomas C. Pochapsky, Brandeis University, Waltham, MA, and accepted by the Editorial Board April 6, 2015 (received for review October 17, 2014) K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) is a on the anionic membrane and that these orientations could be prenylated, membrane-associated GTPase protein that is a critical influenced by the bound nucleotide. Here we present high-resolu- switch for the propagation of growth factor signaling pathways to tion NMR-derived models of the dynamic interactions between diverse effector proteins, including rapidly accelerated fibrosar- K-RAS4B and the lipid bilayer and how they can be impacted by coma (RAF) kinases and RAS-related protein guanine nucleotide disease-associated mutations or interactions with effector proteins. dissociation stimulator (RALGDS) proteins. Gain-of-function KRAS mutations occur frequently in human cancers and predict poor clin- Results ical outcome, whereas germ-line mutations are associated with de- K-RAS4B Activation State Determines Population of Two Major velopmental syndromes. However, it is not known how these Orientations on Lipid Bilayer. Recent developments in solution mutations affect K-RAS association with biological membranes or NMR technology have made it possible to study integral membrane whether this impacts signal transduction. Here, we used solution NMR studies of K-RAS4B tethered to nanodiscs to investigate lipid proteins and those anchored to membranes (7). To overcome the BIOPHYSICS AND

bilayer-anchored K-RAS4B and its interactions with effector protein inherent challenges associated with solution NMR studies of COMPUTATIONAL BIOLOGY 13 RAS-binding domains (RBDs). Unexpectedly, we found that the membrane-associated proteins, we used selective C-labeling of effector-binding region of activated K-RAS4B is occluded by interaction with the membrane in one of the NMR-observable, and thus Significance highly populated, conformational states. Binding of the RAF isoform ARAF and RALGDS RBDs induced marked reorientation of K-RAS4B KRAS (Kirsten rat sarcoma viral oncogene homolog) is frequently from the occluded state to RBD-specific effector-bound states. Im- mutated in pancreatic, colon, and lung tumors, which predicts portantly, we found that two Noonan syndrome-associated muta- poor clinical outcome, whereas germ-line mutations are associ- tions, K5N and D153V, which do not affect the GTPase cycle, relieve ated with developmental disorders, including Noonan syndrome. the occluded orientation by directly altering the electrostatics of two Although K-RAS is an attractive anticancer target, no clinically membrane interaction surfaces. Similarly, the most frequent KRAS successful inhibitors are available. Most disease-associated mu- oncogenic mutation G12D also drives K-RAS4B toward an exposed tations elevate the activated GTP-bound form of KRAS; however, configuration. Further, the D153V and G12D mutations increase the some remain unexplained. KRAS signals from cellular mem- rate of association of ARAF-RBD with lipid bilayer-tethered K-RAS4B. branes; however, our studies revealed that its association with We revealed a mechanism of K-RAS4B autoinhibition by membrane the membrane surface sequesters its binding site for effector sequestration of its effector-binding site, which can be disrupted by proteins, hampering signaling. Some disease-associated KRAS disease-associated mutations. Stabilizing the autoinhibitory interac- mutations disrupt this autoinhibition, identifying a new gain- tions between K-RAS4B and the membrane could be an attractive of-function mechanism and explaining how certain Noonan syn- target for anticancer drug discovery. drome mutations activate K-RAS signaling. Importantly, these findings open new avenues for therapeutic strategies to target KRAS | nuclear magnetic resonance | lipid bilayer nanodisc | oncogenic oncogenic K-RAS through stabilizing autoinhibitory interactions mutation | Noonan syndrome with the membrane.

he K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) Author contributions: M.T.M.-J., C.B.M., M.J.S., G.M.C.G.-S., P.B.S., F.I., L.E.K., B.G.N., and KRAS M.I. designed research; M.T.M.-J. performed research; M.J.S., F.I., and B.G.N. contributed Tprotein product of the gene undergoes posttranslational new reagents/analytic tools; M.T.M.-J., C.B.M., G.M.C.G.-S., P.B.S., L.E.K., and M.I. analyzed farnesylation and C-terminal processing, which, in conjunction with data; M.T.M.-J., C.B.M., and M.I. wrote the paper. a poly-basic hypervariable region (HVR), targets K-RAS4B to The authors declare no conflict of interest. anionic lipid rafts on the intracellular side of the plasma mem- This article is a PNAS Direct Submission. T.C.P. is a guest editor invited by the Editorial brane (Fig. 1A) (1). This localization is essential for K-RAS4B Board. function and enhances signaling fidelity (2). Although the signif- Data deposition: The atomic coordinates and structure factors have been deposited in the icance of membrane tethering of K-RAS4B is well appreciated, a Protein Data Bank, www.pdb.org (PDB ID codes: 2MSC, 2MSD, and 2MSE), and in the high-resolution map of how K-RAS4B interacts with the mem- Biological Magnetic Resonance Bank, www.bmrb.wisc.edu (BMRB entry ID codes: 25114, 25115, and 25116). brane is lacking. Because membrane-anchored RAS presents a 1Present address: Program in Molecular Structure and Function, Hospital for Sick Children, major challenge to crystallization, current structural insights into the Toronto, ON, Canada M5G 1X8. behavior of membrane-anchored RAS have come from a variety of 2Present address: Department of Physiology and Pharmacology, Schulich School of Med- lower-resolution techniques including in vivo FRET-based studies icine & Dentistry, London, ON, Canada N6A 5C1. (3), fluorescence and infrared spectroscopic studies (4–6), and in 3To whom correspondence should be addressed. Email: [email protected]. silico models (3). These pioneering studies suggested that the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. K-RAS4B GTPase domain adopts certain preferred orientations 1073/pnas.1419895112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1419895112 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 A All 11 isoleucines are located <4 Å from the protein surface and are well distributed throughout the protein to provide excellent probes for molecular interactions. To unambiguously identify re- gions of the GTPase domain interacting with or in close proximity to the lipid bilayer, we prepared nanodiscs incorporating lipids + conjugated to the paramagnetic ion gadolinium (Gd3 )and measured broadening of the K-RAS4B Ile Cδ resonances induced by paramagnetic relaxation enhancement (PRE) (10). Mapping the PRE-broadened Ile probes on the structure of K-RAS4B (Fig. 2 A and B and Table S1) revealed that not all of the observed PRE-derived constraints can be simultaneously satisfied by a sin- gle interface, indicative of equilibrium between at least two orientations with distinct sites of membrane association. A comparison of the PRE profiles of the active and inactive forms of K-RAS4B reveals that Ile36 and Ile139 are the residues most sensitive to the activation state (Fig. 2A). Ile36 is located at the C terminus of switch I in close proximity to switch II, and Ile139 is on the opposite side of the GTPase domain in proximity to helix-α4, suggesting that nucleotide loading shifts the equilibrium of membrane association of these two surfaces. To construct structural models of membrane-bound K-RAS4B and identify preferred orientations of the active and inactive forms, we carried out high ambiguity-driven biomolecular docking (HADDOCK) simulations (11) (Table S2) using distance re- B straints derived from the PRE experiments (10) (Fig. 2 A and B). The ensemble of models computed by HADDOCK for activated and inactive K-RAS4B each clearly exhibit two distinct clusters of orientations (Fig. 2C and Fig. S2B), consistent with orientational flexibility of the K-RAS4B GTPase domain (6); however, the relative population of each cluster was dependent on the activation state (Table S2). In the cluster that predominates in the GDP-bound form, most of the K-RAS4B helices are oriented parallel to the membrane, and a surface comprised of the N terminus of β1 (N-β1), α4, β6, α5, and the loop connecting β2 and β3 interfaces with the membrane (Fig. 2 C and D). This surface, located opposite to switch I, is therefore designated the α-interface. By contrast, the helices are oriented semiperpendicular to the membrane in the major cluster for activated K-RAS4B- GMPPNP (a nonhydrolyzable analog of GTP), and the membrane interface is formed by structural elements surrounding C terminus of switch I, including β-strands 1–3oftheGTPase domain β-sheet core, α2, and the C-terminal part of α3(the β-interface). These results are consistent with previous infrared spectroscopy data, which showed that the helical trajectories of K-RAS4B bound to GDP, but not GMPPNP, are mainly parallel with the bilayer plane, although the membrane interfaces were not determined (4, 5). The structural basis for this change in orientation likely in- volves the nucleotide-dependent conformational change in switch Fig. 1. K-RAS4B signaling at the plasma membrane and the nanodisc lipid II, whereby GMPPNP binding both increases the surface-exposed bilayer model for NMR studies. (A) Schematic illustration of K-RAS4B sig- positive charge and decreases the negative charge on the naling on plasma membrane. (Inset) Schematic of a K-RAS4B:nanodisc β D complex. (B) Distribution of eleven isoleucine Cδ throughout the K-RAS4B -interface (Fig. 2 ), electrostatically stabilizing the semiperpen- GTPase-domain. (Right) 1H-13C HMQC spectra of nanodisc-conjugated dicular K-RAS4B orientation (Table S2). Intriguingly, the nucleo- K-RAS4B in the GDP- (Upper) and GMPPNP- (Lower) bound forms. tide-dependent orientation preference of K-RAS4B (Fig. 2 B and C) differs strikingly from that of H-RAS [by molecular dynamics (MD)- and FRET-based studies] (12, 13) and Rheb (by NMR) the Cδ methyl groups (8) of the 11 isoleucine residues in (10), where the activated and inactive forms favor helix-parallel K-RAS4B, which produced excellent quality (signal/noise > 100) and -perpendicular orientations, respectively. Consistent with 1 13 H- C heteronuclear multiple quantum coherence (HMQC) this finding, previous MD simulation studies of K-RAS4B on spectra with 11 well-resolved peaks, in solution (Fig. S1A)and neutral membrane bilayers did not recapitulate the H-RAS– tethered to nanodiscs (Fig. 1B). Tethering was achieved by specific orientations (3). Importantly, the RAS isoforms H-RAS, conjugation of the C-terminal farnesylation site (Cys185) of a N-RAS, and even the splice variant K-RAS4A have C-terminal K-RAS4B variant (C118S) lacking a surface-exposed Cys (Fig. S2A) HVR sequences and lipidation motifs distinct from those of to a thiol-reactive maleimide-functionalized lipid {1,2-dioleoyl- K-RAS4B (1), as well as isoform-specific substitutions in the sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl) C-terminal lobe of the GTPase domain (14), which likely lead to cyclohexane-carboxamide] (PE-MCC)} (9). different orientational equilibria of the GTPase domain.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1419895112 Mazhab-Jafari et al. Downloaded by guest on September 28, 2021 ABmembrane is affected by interaction with the RAS-binding domain (RBD) of the rapidly accelerated fibrosarcoma (RAF) isoform ARAF. Like the RBD of CRAF that is preferentially recruited to K-RAS4B nanoclusters in cells (15), ARAF-RBD contains a positively charged patch adjacent to the RAS-binding site (Fig. S4A) and contains three isoleucine residues well distributed to provide PRE-based restraints. We reconstituted nanodisc-tethered complexes of isotopically labeled K-RAS4B with ARAF-RBD (total molecular weight, ∼200 kDa), performed NMR measurements (Fig. S1B) to deduce PRE distance restraints from the lipid bi- C layer to both K-RAS4B and ARAF-RBD (Tables S1 and S3), and derived HADDOCK models of the membrane-associated complex. Within this complex, the GTPase domain was found in a new semiexposed orientation intermediate between the exposed and occluded orientations (Table S2), which places the cationic surface of the ARAF-RBD in contact with the anionic membrane surface (Fig. 3 A–C and Fig. S4B) and reduces the PRE effect on Ile36, reflecting global reorientation away from the occluded configuration, as well as Ile46, which suggests the β-interface rotates away from the membrane on complex formation. Interest- ingly, the RBD of RAS-related protein (RAL) guanine nucleotide dissociation stimulator (GDS) possesses an anionic surface and D shifts the orientational equilibrium of K-RAS4B away from the occluded orientation toward a fully exposed orientation, as evi- denced by decreased PRE on Ile36 and Ile100, increased PRE on Ile139, Ile142, and Ile163, and only minor PRE perturbations of the RALGDS-RBD resonances, whereas reduced PRE on Ile24 and

Ile46 indicate that K-RAS4B adopts an altered exposed orientation BIOPHYSICS AND

in complex with RALGDS (Fig. S5). These observations strongly COMPUTATIONAL BIOLOGY argue that K-RAS4B interactions with different RBDs can lead to stabilization of distinct GTPase domain/membrane orientations that vary depending on the nature of the effector (Fig. 3C). K-RAS4B complexes with other RBDs remain to be investigated, including the RBD of PI3 kinase, which has been challenging to express in a E soluble form. The probability of forming productive complexes with various effector proteins would be expected to depend on the dynamic conformational equilibrium of K-RAS4B on the membrane and correlate inversely with the transient population of the occluded orientation. Based on the intimate engagement of membranes by K-RAS4B in multiple orientations, we postulate that K-RAS4B signaling output might be altered by mutations that remodel the membrane orientational equilibrium.

Fig. 2. Activated K-RAS4B adopts an occluded orientation on anionic Disease-Associated Mutations Relieve Effector-Binding Site Occlusion. membranes. (A) PRE-induced broadening of K-RAS4B H(13C) resonances by We then sought to address whether oncogenic mutations of Gd3+-conjugated lipid incorporated into nanodiscs. PRE effects detected for KRAS affect the membrane orientation. Generally, oncogenic each isoleucine residue of K-RAS4B presented as ratios of H(13C) resonance K-RAS mutants, such as G12D, are known to increase GTP intensities of K-RAS4B conjugated to nanodiscs in the presence (I*) to those + loading, thus leading to hyperactivation of RAS signaling path- in the absence (Io)ofGd3 . Residues are grouped according to their location ways (16). Here we unveiled that the K-RAS4B G12D mutation with respect to the α and β membrane interfaces defined below. Error bars δ markedly releases the effector-occluded configuration, as evi- based on spectral noise. (B) Map of the PRE effect on K-RAS4B Ile-C . Resi- denced by reduced PRE broadening of Ile21/Ile36 and increased dues broadened more than 20% are highlighted in red and the remainder in A cyan. (C) PRE-driven models of K-RAS4B-nanodisc complexes (see Materials broadening of Ile139/Ile163 (Fig. 4 ), suggesting that oncogenic and Methods for details). (D) Surface electrostatics of K-RAS4B-GDP (Left; mutations could alter the normal function of RAS proteins PDB ID code 4LPK) and K-RAS4B-GMPPNP (Right; modeled from PDB ID code through multiple actions. Gly12 is not spatially proximal to the 3GFT). K-RAS4B is oriented to view the occluded interface from the mem- membrane in either orientation (Fig. 4B); however, the G12D brane. Blue, positive; red, negative. Surface exposed positive (bold) and substitution was previously shown to increase the population of a negative charged residues are indicated. Increased exposure of R102 and nucleotide-free–like conformation of RAS-GTP called state 1, reorientation of E63 contribute to a more positively charged occluded in- which increases the entropy (i.e., mobility) of the switch I/II and terface in the GMPPNP-bound form. (E) Schematic illustration of K-RAS4B α α β helix 3 region (17, 18). The enhanced dynamics in the nucleotide- reorientation on GTPase cycling. The - and -interfaces are shown with a binding region (19) is most likely responsible for destabilizing the helix and an arrow, respectively. β-interface. To further investigate whether the state 1 population influences the membrane orientation, we measured PRE broad- Interaction with RAS-Binding Domains Requires K-RAS4B Reorientation ening of the state 1-selective K-RAS4B mutant V29G (20). The to Expose Effector-Binding Site. To our surprise, the preferred ori- V29G substitution is not proximal to the membrane; nevertheless, entation of activated K-RAS4B sequesters the C terminus of switch I the V29G mutant recapitulated the G12D reorientation toward (i.e., the effector-binding loop) at the membrane surface (Fig. S3) the exposed state (Fig. S6), indicating that the state 1/2 equilibrium and appears incompatible with binding of effector proteins. Thus, plays a role in determining K-RAS4B membrane orientation, ap- we examined how the orientation of activated K-RAS4B on the parently through allosteric modulation of the β-interface (19).

Mazhab-Jafari et al. PNAS Early Edition | 3of6 Downloaded by guest on September 28, 2021 A 1 K5N (21) and D153V (22–24), were shown to induce phosphorylation of MEK1/2 more strongly than WT KRAS; however, the mechanism of their activation has remained elusive. Unlike o oncogenic K-RAS mutations such as G12D, G13D, and Q61H, ARAF-RBD K-RAS4B neither of these germ-line mutations appreciably altered intrinsic 3+ I* / I GTPase activity or nucleotide exchange or sensitivity to GAPs or Gd guanine nucleotide exchange factors (GEFs) in solution; thus, they 0 have been classified as mutants whose phenotypes are not ex- I54 I67 I76 plained by their biochemical properties (25, 26). Intriguingly, we ARAF-RBD find that these mutations change the electrostatic surface of the two main membrane interfaces of K-RAS4B (Fig. 4B). By re- 1 K-RAS4B alone ducing the positive charge on the occluded β-interface and re- K-RAS4B + ARAF-RBD ducing the negative charge on the α-interface of the exposed o orientation, respectively, our models predict that both the K5N

I* / I and D153V mutations would shift the orientational equilibrium of K-RAS4B away from the occluded orientation (Fig. 4 B and E), therefore unleashing the membrane-dependent autoinhibition of 0.5 K-RAS4B signaling. Indeed, PRE experiments demonstrated that I36 I21 I46 I100 I55 I163 I142 I139 I24 I84 I93 both mutations reduced the PRE effect on Ile36 and increased α-interface Remote from that on Ile139 (Fig. 4A), indicating that mutation-induced surface β-interface interfaces electrostatic changes can cause reorientation of K-RAS4B on K-RAS4B Isoleucine anionic membranes. To determine whether the increased pop- B ulation of the binding-competent exposed state enhances interaction of these K-RAS4B mutants with effector proteins, we K-RAS4B used biolayer interferometry (BLI; ForteBio Octet RED96) to directly measure the binding kinetics of K-RAS4B to immobilized ARAF-RBD. We postulate that the degree of exposure of ARAF-RBD the effector binding site will influence the association rate (kON) I36 of the K-RAS4B–effector interaction. Remarkably, the two I139 mutations that most strongly destabilize the occluded orientation, G12D and D153V lead to measurable increases in the I67 association rates (10% and 20%, respectively) of nanodisc- α5 tethered K-RAS4B with ARAF-RBD, whereas the mutations had no significant effect on the association rate of free K-RAS4B C D K167 I54 (Fig. 4 and and Fig. S7). The K5N mutation, which has a more subtle effect on orientation, did not have a detectable K172 effect on the association rates of free or nanodisc-tethered R41 I76 K-RAS4B, but decreased the dissociation rate in a lipid bilayer- K28 K69 K66 independent manner (Fig. 4 C and D). Overall, these disease- associated mutations enhanced the RBD interaction of lipid R68 bilayer-tethered K-RAS4B by 10–25% (Fig. 4D), suggesting that perturbation of the conformational equilibrium contributes C to their increased downstream signaling and is a particularly K-RAS4B GTP ARAF important factor in the case of D153V. To further delineate the RBD GTP K-RAS4B effect of K-RAS4B membrane orientation on RBD binding, we α β β rationally designed a Cys mutation that, on conjugation to α RALGDS PE-MCC, would favor the occluded orientation: A M67C sub- + + RBD stitution introduced on the edge of the occluded interface to avoid spoiling the effector-binding site had no effect on free K-RAS4B binding to immobilized ARAF-RBD. Interestingly, Fig. 3. Occluded K-RAS4B orientation is incompatible with RBD binding. this Cys mutant reduced the affinity of nanodisc-conjugated (A) Effect of interaction with ARAF-RBD on the PRE profile of nanodisc- K-RAS4B twofold (Fig. 4D), presumably by stabilizing the auto- tethered K-RAS4B. (Upper) PRE profile of ARAF-RBD Ile-Cδ resonances in the + + presence of Gd3 -containing (I*) vs. Gd3 -free (Io) nanodisc-tethered K-RAS4B- inhibited occluded conformation through a covalent bond be- GMPPNP. (Lower) PRE profile of nanodisc-tethered K-RAS4B-GMPPNP (red) tween Cys67 and PE-MCC. vs. nanodisc-tethered K-RAS4B-GMPPNP in complex with ARAF-RBD (ma- genta). Residues are grouped according to their location with respect to Discussion the α and β membrane interfaces. (B) PRE-based model of nanodisc-tethered The present data provide, to our knowledge, the first evidence K-RAS4B in complex with ARAF-RBD. The K-RAS4B:ARAF-RBD complex was for release of membrane-dependent autoinhibition as a pre- modeled on the basis of homology to H-RAS:C-RAFRBD (PDB ID code 4G0N) viously unidentified mechanism by which K-RAS4B mutations (37), and this complex was docked to the bilayer surface based on PRE-derived can activate signaling (Fig. 4E). The extent to which orienta- constraints using HADDOCK. The lowest HADDOCK-scored structure within tional effects contribute to K-RAS4B G12D signaling in the cell the major cluster is shown. (C) Schematic illustration of membrane orientations of K-RAS4B complexed with RBDs of ARAF vs. RALGDS. relative to effects on the GTPase cycle remains to be investigated; however, the K5N and D153V Noonan/CFC syndrome mutations suggest that perturbing membrane orientation to un- Our model further predicts that K-RAS4B signaling could leash autoinhibition may alone be sufficient to produce a disease be modulated by mutations that directly alter the membrane phenotype. Importantly, KRAS K5N and D153V mutations have interaction surfaces. Two germ-line KRAS mutations identified also been detected in lung, stomach, and renal cancers (27, 28), in Noonan and cardio-facio-cutaneous (CFC) syndrome patients, whereas germ-line and somatic mutations encoding K5E were

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1419895112 Mazhab-Jafari et al. Downloaded by guest on September 28, 2021 A K-RAS4B The oncogenic and RASopathy-associated K-RAS4B muta- 1 K-RAS4B K5N tions examined here relieve a membrane-associated orientation K-RAS4B D153V that occludes effector binding, which may define a new mecha- K-RAS4B G12D nism by which KRAS mutations can enhance signaling (Fig. 4E). o The K-RAS4B orientational equilibrium may further be modu- 2+ I* / I lated by (i) protein–protein interactions such as Ca /calmodulin binding to the HVR (1), (ii) posttranslational modifications such as PKC phosphorylation of Ser181 in the HVR (1) and acety- lation or monoubiquitinylation of K104 (1) in the β-interface, or 0.5 (iii) alterations of the membrane lipid composition. Further I36 I21 I46 I100 I55 I163 I142 I139 I24 I84 I93 studies are needed to elucidate how the intimate communication α-interface Remote from β-interface between RAS and membrane may be dynamically regulated by these K-RAS4B Isoleucine interfaces regulatory factors. Interestingly switch II is exposed in the major I36 conformation of the inactive GDP-bound form of K-RAS4B, sug- B Occluded Exposed gesting it is accessible to activation by RAS GEFs. G12 Finally, K-RAS4B, a well-validated oncogenic driver for many cancer types, is a challenging drug target (16). Although some lead I139 G12 K5 D153 compounds have been identified (31), there are still no clinically effective RAS inhibitors available. The propensity of K-RAS4B α4 β1 to associate with the membrane in a manner that occludes its I139 α5 β2 effector-binding site may reveal a novel and unexplored therapeutic C α5 β3 – β6 I36 target at the protein membrane interface. Our model suggests that pharmacological modulation of the orientational equilibrium may C K5 D153 be exploited to sequester activated K-RAS4B mutants.

C 1 1 1 Materials and Methods

d Preparation of proteins and nanodisc-tethered protein complexes, NMR ON K OFF BIOPHYSICS AND k 0 k 0 0 measurements, and BLI assays are fully described in SI Materials and Methods. COMPUTATIONAL BIOLOGY

WT NMR- and PRE-Guided Molecular Docking Simulations. All docking simulations WT WT K5N K5N K5N G12D G12D M67C G12D were performed using HADDOCK 2.0 (11, 32) as described previously (10). M67C M67C D153V D153V D153V Derivation of the full-length K-RAS4B and nanodisc models are described D 2 1 1 in SI Materials and Methods. The K-RAS4B: nanodisc [MSP1D1, 80% 1,2- d K

ON 1 dioleoyl-sn-glycero-3-phosphocholine (DOPC), 20% 1,2-dioleoyl-sn-glycero- k OFF L 0 k 0 0 3-phospho- -serine (DOPS)] complex was then subjected to energy minimization in CNS before HADDOCK simulations. HADDOCK ambiguous

WT restraints were generated from the PRE measurements of K-RAS4B tethered WT WT K5N K5N K5N 3+ G12D G12D M67C G12D

M67C to Gd -containing nanodiscs. Because the lateral diffusion rate of DOPC at M67C D153V D153V D153V room temperature (∼8.2 × 103 nm2/ms) (33) is high relative to T2 relaxation E GTP GTP GTP times of macromolecules (e.g., nanodisc-tethered Rheb ∼30 ms), an as- G12D + +1 sumption was made that the paramagnetic Gd3 ion conjugated to 1,2- K5N D153V -1 distearoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepenta- -1 acetic acid (gadolinium salt; PE-DTPA) uniformly sampled all positions on the nanodisc surface (78 nm2) during the time course of the measurement. Thus, ambiguous restraints were generated between PRE-affected isoleucine residues on K-RAS4B and any lipid headgroup atom on the nanodisc surface. More Effector-Accessible States Isoleucine residues exhibiting peak broadening of >20% were defined as active, and the adjacent N- and C-terminal residues were defined as passive Increased RAS Signaling residues. For simulation of K-RAS4B-GDP, isoleucines 24, 46, 55, 100, 139, 142, and 163 were defined as active and residues 23, 25, 45, 47, 54, 56, 99, Fig. 4. K-RAS4B oncogenic and Noonan syndrome mutations relieve the oc- 101, 138, 140, 141, 143, 162, and 164 were defined as passive. For simulation cluded orientation and alter RBD binding kinetics on lipid bilayer. (A) PRE profiles of K-RAS4B-GMPPNP, isoleucines 24, 36, 46, 55, 100, 139, 142, and 163 were of WT K-RAS4B vs. K5N, D153V, and G12D mutants. 1H-13CHMQCspectraare identified as active residues and 23, 25, 35, 37, 45, 47, 54, 56, 99, 101, 138, shown in Fig. S1. Error bars based on spectral noise. Residues are grouped 140, 141, 143, 162, and 164 as passive residues. Surface electrostatics of according to their location with respect to the α and β membrane interfaces. K-RAS4B were generated by the Poisson-Boltzmann Solver (34–36). The (B) Location of mutations and Ile36/Ile139 probes on exposed and occluded K-RAS4B:ARAF–RBD complex was modeled by assembling the K-RAS4B model

K-RAS4B models. (C and D) Relative association rates (kON), dissociation rates described above with the NMR structure of ARAF-RBD [Protein Data Bank (kOFF), and dissociation constants (Kd)for(C)freeK-RAS4Band(D)nano- (PDB) ID code 1WXM], guided by the H-RAS:C-RAFRBD crystal structure (PDB disc-tethered K-RAS4B binding to ARAF-RBD. Binding kinetics of K-RAS4B ID code 4G0N) (37). In simulations of the complex, isoleucines 24, 36, 46, 55, (analyte) to immobilized ARAF-RBD (ligand) was measured by biolayer 100, 139, 142, and 163 of K-RAS4B and isoleucines 54 and 76 of ARAF-RBD

interferometry (Fig. S7). Each kON and kOFF rate was determined using three were identified as active residues, and the flanking residues were selected as concentrations of K-RAS4B. Error bars represent SD. Kd values were passive. To normalize the contribution of both molecules to the K-RAS4B: determined from kON and kOFF, and SD was propagated accordingly. ARAF-RBD complex models, ARAF-RBD restraints were weighted more (E) Schematic illustration of K-RAS4B reorientation induced by mutations. The heavily to compensate for fewer Ile probes. Ambiguous distance restraints + mutation locations are shown by cyan spheres and changes in net charge are were assigned a range of 2–5 Å. Although the paramagnetic effect of Gd3 depicted by ±1. The wavy arrow represents the increased dynamics of the can extend to a 20-Å radius (38), each lipid headgroup on the membrane + β-interface associated with the state 1 population of the G12D mutant. The surface is only partially (2.5%) occupied by Gd3 -conjugated lipid; thus, a curved green arrow represents the reorientation. reduced upper limit of 5 Å was estimated. An upper limit of 2 Å was selected for isoleucines broadened >50%, as well as K-RAS4B Cys185. Additionally, the Lys175 at the beginning of the polybasic region was identified as an recently identified in CFC syndrome (29) and chronic myelomono- active residue. The CNS topology and the parameter files for GDP, GMPPNP, cytic leukemia, respectively (27, 30). DOPC, and DOPS were generated using the HIC-UP server (39) and modified

Mazhab-Jafari et al. PNAS Early Edition | 5of6 Downloaded by guest on September 28, 2021 according to the data reported for these small molecules in the Automated algorithm (41), setting an RMSD cutoff of 7.5 Å (11, 32) and cluster size Topology Builder (ATB) and Repository (40). The docking protocol was composed cutoff of 20 structures. The HADDOCK scores of all 200 structures were of a 3,000 rigid-body docking stage, where the top 200 ranked structures based plotted against RMSD relative to the global mean structure, defined as the on the HADDOCK scores were refined using semiflexible simulated annealing, solution with the lowest average pairwise RMSD to all other 199 solutions. followed by water refinement. The docking protocol was executed with default All structural manipulations and measurements in 3D space were performed HADDOCK script parameters with an additional Powell energy minimization using CNS (42). step of the lipid headgroups before the semiflexible refinement stage. ACKNOWLEDGMENTS. We thank Sharon Campbell (University of North Cluster Analysis of HADDOCK Models. Pairwise backbone RMSD values were Carolina at Chapel Hill) for providing the H-RAS resonance assign- tabulated for the K-RAS4B G-domain (residues 4–171) after alignment of the ments, Bob Berno (McMaster University) for 31P NMR data collection, nanodiscs within the same plane and a translational and 360° rotational (in and Trevor Moraes and Anastassia Pogoutse (University of Toronto) for the 5° increments) RMSD minimization search that were confined only to use of BLI instrumentation. This work was supported by Cancer Research So- movements within the 2D plane of the membrane surface, as described ciety (Canada) Grant 14014, the Princess Margaret Cancer Foundation, and previously (10). The K-RAS4B:nanodisc position in each solution was kept Canadian Cancer Society Grant 703209. NMR spectrometers were funded by constant during pairwise RMSD calculation. Cluster analysis was performed the Canada Foundation for Innovation. L.E.K., B.G.N., and M.I. hold Canada on the calculated pairwise RMSD values using a previously described Research Chairs.

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